Metabolism of phenylbenzoate by Pseudomonas sp. strain TR3

Thomas Reich, Stefan Schmidt *, Peter FortnagelAbteilung fu«r Mikrobiologie, Institut fu«r Allgemeine Botanik der Universita«t Hamburg, OhnhorststraMe 18, D-22609 Hamburg, Germany

Received 25 January 1999; received in revised form 21 May 1999; accepted 27 May 1999

Abstract

A bacterial isolate, tentatively identified as Pseudomonas sp. strain TR3, was found to utilize the diaryl ester phenylbenzoateas sole source of carbon and energy. This strain has the ability to productively degrade phenylbenzoate and some substitutedderivatives by a catabolic sequence which was characterized biochemically. The biodegradation of phenylbenzoate is thusinitiated by an inducible esterase, effectively hydrolyzing the diaryl esters to produce stoichiometric amounts of twomonoaromatic metabolites, identified as benzoate and phenol in the case of phenylbenzoate. The diaryl ester p-tolylbenzoatewas hydrolyzed to yield benzoate and 4-methylphenol while 4-chlorophenylbenzoate gave rise to the production of benzoateand 4-chlorophenol. These monoaromatic catabolites were further degraded via the oxoadipate pathway. ß 1999 Federationof European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Biodegradation; Diaryl ester; Phenylbenzoate; Hydrolysis ; Ortho pathway; Pseudomonas

1. Introduction

Ester bonds are a structural feature present in alarge number of industrially manufactured organiccompounds of environmental concern such aspolymers, solvents, pharmaceutical and medicalproducts and pesticides [1,2]. During the past decade,the diaryl ester phenylbenzoate was produced on anindustrial scale [2], an important area being the pro-duction of liquid-crystalline polymers (e.g. used inLCD-displays) [3]. Basically, these thermotropic

liquid-crystalline polymers consist of benzene ringsinterconnected by ester linkages [4].

Although a great number of bacteria with the abil-ity to hydrolyze ester bonds present in environmen-tally relevant compounds has been reported [5^7], tothe best of our knowledge, no information is avail-able concerning the bacterial degradation of phenyl-benzoate, the basic structure present in certainliquid-crystalline polyesters.

2. Materials and methods

2.1. Isolation and identi¢cation of the organism

Pseudomonas sp. strain TR3 was isolated from en-richment cultures inoculated with soil samples (with-out a known history of pollution) collected in the

0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 2 7 5 - X

* Corresponding author. Tel. : +49 (40) 82282463;Fax: +49 (40) 82282509;E-mail: stefan.schmidt@mikrobiologie.uni-hamburg.de

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Hamburg area, by originally using xanthone as thesole source of carbon and energy. The characteriza-tion of the isolate was done according to standardprocedures [8^10].

2.2. Media and culture conditions

Growth of the organism in liquid culture was per-formed using a known mineral medium [11] contain-ing phenylbenzoate or its derivatives at a ¢nal con-centration of 25 mM. Cells were grown in 1-lErlenmeyer £asks closed (not gas-tight) with te£on-sealed screw-caps, ¢lled with 50 ml of the ¢nal me-dium and incubated at 28³C on a rotary shaker at100 rpm. Cell growth was monitored by measuringthe absorbance at 578 nm using a Uvicon 922(Kontron, Mu«nchen, Germany) spectrophotometer.For the growth of Pseudomonas sp. TR3 with anon-aromatic organic compound, acetate was addedto the mineral medium to a ¢nal concentration of2.5 mM.

2.3. Preparation of crude extracts

Cells were harvested by centrifugation at10 000Ug for 15 min at 4³C and subsequentlywashed twice with phosphate bu¡er (27 mM, pH7.2). The cells were disrupted using a French pressat 10 000 psi and the cell lysate was centrifuged at40 000Ug for 60 min. The supernatant was collectedand used for enzyme assays. Soluble protein wasdetermined according to Bradford [12] using BSAas standard.

2.4. Measurement of oxygen uptake

Exponentially growing cells were harvested,washed twice with phosphate bu¡er and resuspendedin phosphate bu¡er (27 mM, pH 7.2) to an opticaldensity at 578 nm (OD578) of about 0.9. Oxygenuptake rates were measured using a Clark-type oxy-gen electrode as described [13].

2.5. Analytical methods

Qualitative and quantitative HPLC analysis wasperformed as described [13], using an Inertsil ODS-3 column (5 Wm, 250 mm by 4.0 mm, GL Science,

Schambeck, Bad Honnef, Germany). The mobilephase consisted of methanol-water (70:30, v/v),acidi¢ed with 1 g l31 o-phosphoric acid. For thequanti¢cation of phenylbenzoate present in bacterialcultures, replicate £asks were extracted at timed in-tervals with 0.1 volume of chloroform and the diarylester was quanti¢ed by HPLC as reported [13]. Therecoveries were found to be quantitative in a rangeof 0.1^1.000 mg of phenylbenzoate per litre medium.

3-Oxoadipate was detected according to the meth-od of Friedemann and Haugen [14] by means of thinlayer chromatography (TLC) of the dinitrophenylhy-drazon derivatives. Results were con¢rmed by com-parison with authentic standards using TLC plates(silica gel G plates, Merck, Darmstadt, Germany)and a solvent system consisting of 2-propanol-am-moniak-water (20:1:2, v/v/v).

2.6. Enzyme assays

The esterase activities were assayed by high per-formance liquid chromatography (HPLC) using areaction mixture containing in a ¢nal volume of1 ml, 970 Wl phosphate bu¡er (27 mM, pH 7.2),substrate (2.5 Wmol) and an appropriate amount ofprotein (1^15 Wg ml31). These assays were started byaddition of the substrate and incubated at 30³C for15 min. Every minute, 100 Wl was withdrawn and thereaction was stopped immediately by adding anequal volume of o-phosphoric acid. After centrifuga-tion, the supernatant was analyzed by HPLC. Allassays were performed in triplicate. In addition, theesterase activity was followed by monitoring the ab-sorption increase due to the production of p-nitro-phenol from the general esterase substrate p-nitro-phenylacetate at 405 nm. This assay was carriedout at 30³C in a ¢nal volume of 1 ml containing965 Wl phosphate bu¡er (27 mM, pH 7.2), p-nitro-phenylacetate (2.5 Wmol) and enzyme solution (pro-tein 1^15 Wg ml31).

Benzoate 1,2-dioxygenase and phenolhydroxylaseactivities were measured employing whole cells asdescribed [15,16]. Reactions were stopped with liquidnitrogen. Samples were centrifuged and the superna-tant was analyzed by HPLC. Catechol 1,2-dioxyge-nase (C12O) and catechol 2,3-dioxygenase (C230)were assayed using crude extracts by following pre-viously described procedures [17,18].

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2.7. Chemicals

Phenylbenzoate and benzoate were purchasedfrom Aldrich (Steinheim, Germany). Benzylben-zoate, p-tolylbenzoate, 4-chlorobenzylbenzoate and4-chlorophenylbenzoate were obtained from Lancas-ter (Mu«hlheim, Germany), whereas 3-oxoadipatewas from Sigma (Deisenhofen, Germany). All otherchemicals used were of the highest purity commer-cially available.

3. Results and discussion

The bacterial strain employed was originally ob-tained from batch enrichment cultures using soil

samples as inoculum and xanthone as sole sourceof carbon and energy. After several transfers, thestrain TR3 was isolated and tentatively identi¢edas a Pseudomonas species on the basis of the follow-ing morphological and physiological characteristics.It was a strictly aerobic, Gram-negative, oxidase andcatalase-positive rod (about 2.0 by 0.7 Wm). Capsulesor spores were not formed. A single polar £agellumwas present. For additional criteria see Table 1.

The ability of Pseudomonas sp. strain TR3 to growwith phenylbenzoate as only source of carbon andenergy is shown in Fig. 1. Growth, expressed as theincrease in OD, was correlated with the removal ofup to 25 mM phenylbenzoate from the cultures.Heat-inactivated cells (incubated for 15 min at70³C) used as controls showed no signi¢cant abioticloss of phenylbenzoate. In the absence of phenylben-zoate, no production of biomass was detected. Thedoubling time of the bacterium during the exponen-tial growth phase at 28³C was determined as about3 h. During growth, two metabolites transiently ac-cumulated which we identi¢ed by HPLC as phenoland benzoate (Fig. 1). In addition to phenylben-

Table 1Basic morphological and physiological characteristics of Pseudo-monas sp. TR3

Characteristic Result

Mol% G+C of DNA 67Cell shape Rod (about 2.0U0.7 Wm)Motility + (Single polar £agellum)Gram stain NegativeCatalase +Oxidase +Growth at 4, 20, 28 and 37³C +Pigment productionFluorescein +Pyocyanin 3OF test +Indole 3Starch hydrolysis +Hydrolysis of gelatin +Hydrolysis of Tween 80 3Denitri¢cation +Arginine dihydrolase +Assimilation ofMannite 3L-Arabininose 3D-Mannose 3Lactate 3Citrate 3Glucose +Acetate +Xanthone +Phenylbenzoate +p-Tolylbenzoate +p-Chlorophenylbenzoate +Phenol +Benzoate +Catechol +

Fig. 1. Growth of Pseudomonas sp. strain TR3 with phenylben-zoate as the only carbon source. Growth of the culture (F) andthe concentration of phenylbenzoate (b) are plotted against thetime together with controls in the absence of phenylbenzoate (E)and controls using heat-inactivated inocula in the presence ofphenylbenzoate (a). The transient production of phenol (7) andbenzoate (P) was determined by HPLC. All values are means(n = 3) with a S.D.97%.

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zoate, our strain was able to grow with p-tolylben-zoate (td = 7.5 h) and 4-chlorophenylbenzoate(td = 9 h).

Crude extracts were used to verify whether thedegradation of these compounds was initiated byhydrolysis of the ester bond as implied by the tran-sient formation of phenol and benzoate in culturesgrowing with phenylbenzoate. Using crude extractsfrom phenylbenzoate-grown cells, we were able toshow the stoichiometric release of phenol and ben-zoate from this diaryl ester (Fig. 2A). In the case of4-chlorophenylbenzoate, stoichiometric amounts of4-chlorophenol and benzoate (Fig. 2B), as well as4-methylphenol and benzoate in the case of p-tolyl-benzoate (Fig. 2C), were produced from the diarylesters. Biodegradation of phenylbenzoate and deriv-atives is thus initiated by the activity of an inducibleesterase, e¡ectively hydrolyzing the diaryl ester toproduce two monoaromatic metabolites. Crude ex-

Table 2Speci¢c oxygen uptake rates for phenylbenzoate, derivatives anddegradation metabolites by resting cells of Pseudomonas sp. strainTR3

Substrate Speci¢c activitya aftergrowth with

Phenylbenzoate Acetate

Phenylbenzoate 259 6 5p-Tolylbenzoate 307 6 54-Chlorophenylbenzoate 303 6 54-Chlorobenzylbenzoate 66 6 5Benzylbenzoate 252 6 5Phenol 52 6 54-Chlorophenol 54 6 54-Methylphenol 53 6 5Benzoate 301 6 5Catechol 1 090 42Acetate 30 337

aSpeci¢c oxygen uptake rates are given in nmol O2 consumedper minute per mg of protein and are corrected for endogenousrespiration. All data shown are the means of at least three inde-pendently performed experiments.

6Fig. 2. Hydrolysis of phenylbenzoate and derivatives by crude ex-tracts obtained from phenylbenzoate-grown cells of Pseudomonassp. TR3. Shown is the production of monoaromatic metabolites(P= phenol�a�/4-chlorophenol�b�/4-methylphenol�c�,7= benzoate�aÿc�) from phenylbenzoate (a), chlorophenylben-zoate (b) and p-tolylbenzoate (c).

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tracts obtained from acetate-grown cells showed nosigni¢cant hydrolysis of these diaryl esters (data notshown).

Results obtained from oxygen uptake measure-ments clearly indicated that the enzyme activities re-sponsible for the oxidation of phenylbenzoate, someof its derivatives and structurally related compoundssuch as benzylbenzoate were induced by growth withphenylbenzoate. A remarkably high oxygen con-sumption was detected in the presence of catechol,making phenylbenzoate metabolism via this well-known intermediate of many biodegradation path-ways involving aromatic pollutants very likely. Ace-

tate-grown cells were not induced for the oxidationof phenylbenzoate or the other aromatic compoundstested (Table 2). High esterase activities were de-tected in cell free extracts from phenylbenzoate-grown cells, whilst extracts from acetate-grown cellscontained no signi¢cant activity (Table 3). In addi-tion, the data obtained indicate that phenylbenzoateinduces a hydrolase rather speci¢c for diaryl esters.

The induction of catabolic enzymes required forthe degradation of the above-mentioned metabolitesphenol and benzoate after growth with phenylben-

Table 3Speci¢c esterase activities in crude extracts of Pseudomonas sp.TR3

Substrate Speci¢c activitya aftergrowth with

Phenylbenzoate Acetate

Phenylbenzoate 218 6 5p-Tolylbenzoate 207 6 54-Chlorophenylbenzoate 117 6 5Benzylbenzoate 185 6 54-Chlorobenzylbenzoate 126 6 5p-Nitrophenylacetateb 154 6 5

All data shown are the means of at least three independently per-formed experiments.aSpeci¢c activities (nmol min31 mg31 protein).bEnzyme activities using p-nitrophenylacetate as substrate wereassayed spectrophotometrically.

Fig. 3. A proposed pathway for the biodegradation of phenyl-benzoate by Pseudomonas sp. strain TR3. The diaryl ester phen-ylbenzoate is hydrolyzed by an inducible esterase to yield phenoland benzoate. These two compounds are channelled via the cen-tral pathway intermediate catechol into the ortho pathway.

Table 4Activities of catabolic enzymes in crude extracts of Pseudomonassp. strain TR3

Enzyme and substrate Speci¢c activitya aftergrowth with:

Phenylbenzoate Acetate

Benzoate 1,2-dioxygenaseBenzoate 101 6 54-Chlorobenzoate 44 6 54-Methylbenzoate 67 6 5PhenolhydroxylasePhenol 33 6 54-Chlorophenol 9 6 54-Methylphenol 17 6 5Catechol 1,2-dioxygenaseCatechol 433 6 54-Chlorocatechol 40 6 54-Methylcatechol 57 6 5

aSpeci¢c activities are given in nmol min31 mg31 protein. Alldata shown are the means of at least three independently per-formed experiments.

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zoate is demonstrated in Table 4. Phenylbenzoate-grown cells exhibited signi¢cant benzoate 1,2-dioxy-genase and phenolhydroxylase activities as well as ahigh activity of the catechol 1,2-dioxygenase, fur-nishing the corresponding muconic acids. Again, noinduction of these enzymes was detectable in crudeextracts from acetate-grown cells.

A pathway for the degradation of phenylbenzoateby Pseudomonas sp. strain TR3 is proposed on thebasis of our results (Fig. 3). Upon attack of an in-ducible diaryl ester hydrolase, the ester-bridge iscleaved yielding phenol and benzoate. These twomonoaromatic products are catabolized with theaid of the corresponding enzymes of the ortho path-way via 3-oxoadipate as reported [19].

Our results concerning the ability of Pseudomonassp. strain TR3 to e¡ectively degrade selected diarylesters does indicate that liquid-crystalline polymersor at least the basic structures, might be degradable.However, more research is required to evaluate theimpact of substituents on the biodegradability ofthese diaryl esters.

Acknowledgements

This research was supported by the Bundesminis-terium fu«r Bildung, Wissenschaft, Forschung undTechnologie (BMBF Grant number 0318896 C).

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